JP2002517212A - Analysis method using multiple labeled viruses - Google Patents

Abstract

The present invention relates to a method for detecting a target material in a sample, which method comprises the steps of: a) exposing a sample, expected to contain that target material, to at least two viruses which are capable of binding directly or indirectly with that target material so as to form a virally bound target material and to endow the virally bound target material with a distinctive property; and b) cultivating the product from stage a in the presence of an indicator material to which the viruses carried by the virally bound target material attach so as to cause the indicator material to adopt the distinctive property of the virally bound target material; and c) monitoring the presence or otherwise of virally attached indicator material. The invention also provides a kit for use in the method of the invention.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention is a method for detecting a substance, in particular two or more viral components that confer specific properties on the substance to be detected by the presence of the substance in the sample to be verified. To the substance.

2. Prior Art Many sample analysis techniques exist for detecting substances present in a sample. For example, chemical analysis, chromatography techniques, and spectroscopy techniques can be used to identify individual chemical molecules. However, such a technique cannot easily identify a living body in a sample. Nucleic acid methods such as deoxyribonucleic acid hybridization (DNA) are used for DNA and ribonucleic acid (RN).
Used to detect A). However, these methods lack sensitivity. The nucleic acid target can be amplified and detected by using an amplification method such as the polymerase chain reaction (PCR). However, this method is complicated and expensive, and is specified only for nucleic acid detection. By using an immunological method such as Western blotting, enzyme immunoassay (EIA), or enzyme-linked immunosorbent assay (ELISA), a specific molecule such as a protein can be detected. However, these methods have a detection limit from picomoles per liter (pmol) to subpicomoles, and are therefore unsuitable when only a small amount of the substance to be detected is present. In order to improve the sensitivity of such detection methods, radioactive substances,
Labeling with enzymes, gold particles or bacteriophages has been proposed. However, these proposals tended to be unclear because the labeled attachment to the target or other substances in the sample to be evaluated were unspecified.

[0003] For example, PCT Application No. WO 92/02633 discloses that bacteria in a sample are treated in a first culture stage with a specific virus or phage that infects the bacteria that are likely to be present in the sample. We propose a way to identify. The infected sample is processed to kill or eliminate exogenous phage particles that are not infected with bacterial cells, ie, are protected within the cells. The resulting bacterial cells are cultured in a second culture stage to replicate and lyse the phage particles protected in the infected bacterial cells. This releases a new generation of phage particles into the culture medium. This can be detected by performing a second culture stage in the further presence of the first bacterial cell and by detecting dead bacterial cells that are formed together with the replication of the virus particles in the infected cells. Can be. In addition, proteins released by lysis of virus particles can be detected by protein staining or other methods. If the expected bacteria are present in the first sample, the viral particles will be present in the infected bacterial cells from the first culture of the sample and will be found in the second culture stage. However, if the sample does not contain the expected bacteria, the virus will not replicate and lyse in the second culture stage since the cells of the sample will not be infected in the first culture stage. This technique is known as phage amplification.

[0004] A modified version of the phage amplification method disclosed in PCT Application No. WO 97/22713 uses a second culture stage in the presence of bacteria that have a higher replication rate than those used in the first culture stage and are more susceptible to infection. Is performed. In this way, slowly growing bacteria, such as Mycobacterium tuberculosis, can be rapidly detected by the effect of promoting the second culture stage. However, both variants of these phage amplification methods require a virus that is capable of infecting specific and expected bacteria, which strictly limits the range of distinguishable substances and the range of virus particles that can be used. Restrict to It is desirable not only to detect a particular organism in a sample, but also to detect various other substances in the sample. For example, where it may affect the user or the environment,
It is desirable to detect heavy metals and other contaminants in water and waste to determine if one or more substances interact with each other. That is, it is desired to provide a simple and effective method for knowing the influence of a medicine or a drug on a living body or a medical condition requiring treatment with a medicine or a drug. In in vitro homogeneous immunoassay methods, scintillation proximity and fluorescence polarization / fluorescence quenching are used to report molecular interactions in screening assays. Performing these tests is quick and simple, but generally insensitive. As a Vivo Assay System,
A yeast two-hybrid system is used that exhibits molecular interactions in cells, where the interacting proteins act as promoters for gene transfer. However, this protein / protein interaction is disrupted by the drug at the time of the assessment. The yeast two-hybrid system also requires the synthesis of the test components in yeast cells. This limits the range of molecular interactions that can be examined.

[0005] The method devised below is a detection method that is applied to detect various substances and products resulting from the interaction of the substances, and has a wide application range. In the method of the present invention,
Various marker substances can be used to identify the substance to be detected, giving the user flexibility in the substance used. In the method of the present invention, two different virus particles or active ingredients of the virus are:
It is directly or indirectly attached as a tag to the substance to be detected, and is referred to as a target substance hereinafter. These tags provide the target material with unique properties that individually distinguish the tag material from any of the properties of the virus particle. The tagged material is then examined for the presence or absence of a distinguishable property by culturing the tagged material in the presence of bacteria capable of infecting the two viruses. The virus referred to here is a pure virus and a living body that infects bacteria as well as a pure virus. That is, the term virus includes: a. A component of a virus that has virus-derived characteristics. b. A packaged phagemid that can grow as a plasmid in a bacterial host, with a cross between the plasmid and the virus. These are wrapped and hidden like virus particles by the presence of helper virus, but cannot independently produce viral progeny. c. A virus that is lytic to bacteria, grows and replicates in the bacterium without lysis of the bacterium capable of growing and replicating, and produces offspring. If the bacterial cell is infected with more virus than necessary to reach the area between the infecting virus and the infectious bacterial cell,
The bacterial cell is not infected by one or more virus particles. The amount that such double infection occurs sufficiently and does not impair the result of the test of the present invention can be statistically calculated, and is hereinafter referred to as a statistical amount. This statistical calculation can be obtained by simple tests and error tests.

In the method of the present invention, since two virus particles are physically linked via a target substance, each virus particle infects a bacterial cell, and the cell has both specificity of the infecting virus. Properties are given. Bacterial cells infected with both viruses and having two specific properties are easily distinguished from cells having only one of these properties. For example, infected cells can be cultured in situations where only one of these properties survives, for example, certain antibiotics or certain temperature or pH conditions. Infected cells with both these properties survive and replicate. If a lytic virus is used as a tag on a target material, the virus replicates in infected bacterial cells,
It also produces progeny virions that are released to initiate the infection and replication cycle. That is, in the presence of the tagged target material, a cascade of bacterial growth indicates that the target material is present in the initial sample. If no tagged material is present at the culture stage, little or no bacteria will grow.

DISCLOSURE OF THE INVENTION Accordingly, the present invention provides a method for detecting a target substance in a sample. The method comprises the following stages. a) direct or indirect binding of a sample suspected of containing the target substance to the target substance in order to form a virus-bound target substance and obtain a virus-bound target substance having specific properties; B) exposing the product from stage a to the virus-bound target in the presence of the indicator, preferably in the presence of more than the statistical amount of the indicator. Incubate the virus carried by the attachment of the virus-bound target material so that it can accept the specific properties of the material, and c) monitor the presence or absence of the indicator attached to the virus.

The present invention can be applied to a sample in which a target substance is present, or a sample produced by the interaction of other components in the sample or the interaction of a component of the sample with an external component added thereto. . For convenience, the vocabulary of a sample containing a target material is generally used to indicate a material that initially contains the target material or a material from which the target material was produced by some mechanism. The method of the present invention can be used for potency assays of the presence of a target substance in a sample and its approximate mass or concentration, and not only detects the presence or absence of the target substance, but also the amount of the target substance. May be used for dynamic detection. For example, by monitoring the development of secondary characteristics of the stage b product, such as color, the occurrence of the target material in the sample can be monitored. For convenience, the vocabulary of detection as used herein refers to an assay of both the nature and mass of the target substance in a sample, wherein the detection is performed intermittently or continuously for a period of time,
Alternatively, it is performed as one observation. Preferably, the culturing stage b is performed in a state where the indicator attached with the virus survives, but the indicator only attached with the virus or not attached at all is not alive.

As mentioned above, at least two different viruses need to be attached to the target material, and then the stage b culture condition is selected such that only the indicator having the properties conferred by both viruses will survive can do. Commonly used viruses are of different types, and each virus imparts different properties to the virus binding target material, ie, the indicator. However, the use of homologous viruses and the introduction of desirable components not normally present in the virus in a well-known manner and mutating the virus to confer its properties on the virus used in the method of the invention Are also within the scope of the present application. For example, a virus can be treated in a conventional manner to introduce a factor type that confers resistance to a particular antibiotic. This may be done for each virus bound to the target material. Such mutations can introduce other properties into the virus, such as heat or light sensitivity, and the conditions under which cultivation in stage b produces or reflects various properties of the indicator. be able to. As described above, a virus is bound to a target substance and imparts at least two or more properties to the target substance. If desired, additional properties of the virus or additional virus may be imparted to the target material to allow or facilitate detection of the indicator attached to the virus. For example, a third virus that confers photoluminescent properties to the virus-bound target material can be bound to the target material.
Therefore, the presence of the target substance bound to the virus can be easily detected by illuminating the product of stage b with UV, IR or visible light and causing the substance having the third virus to fluoresce. Alternatively, this property may be used as an indicator, for example, β
-An indication of an enzyme not normally represented by galactosidase, luciferase or alkaline phosphatase.

[0010] For convenience, the present invention provides a virus mutated to have one factor type that confers resistance to antibiotic A and a mutation mutated to have another factor type that confers resistance to antibiotic B. This will be described from the viewpoint of binding another virus. The target material may be a virus, a bacterium, a protozoan, a eukaryotic cell, a fungus or a part of another organism, or a part of an organism. However, this target substance is a residue of a living body, for example, a protein, hormone, nucleic acid or acid sequence hidden or generated by the living body, a prion or an antibody. Alternatively, as described above, the target substance may be a drug, for example, a medicament or a biocide, such as a biocide such as a peptide acid, a bactericide, etc., whose efficacy may be against a particular organism. For the detection of Or, a drug produced by the interaction of two other substances, such as hormones and proteins, which, when present in a sample, prevent the substance from producing that hormone or protein, or a substance that is detected for promotion. Can measure the effectiveness of Also, the presence of impurities or contaminating organisms or inorganic chemicals, for example, in water or products, can cause health and other harmful factors. Because of the variety of components that can be tagged by a virus, the present invention is applicable in a variety of situations where it is necessary to detect very small amounts of a substance, typically less than 100 parts per million. is there.

The target substance is ideally presented at two sites where the virus can bind directly to the target substance. Such binding of the target substance can be directly to the virus, for example, at a suitable site on the surface of the virus particle or at a site mutated in a known manner that binds to the target substance. However, either or both of the virus particles may be bound by an intermediate binding substance or ligand, such as a protein, amino acid, lectin, peptide, antibody, monoclonal antibody, nucleic acid or other substance that recognizes a different region of the target substance. Binding to a target material is also within the scope of the present invention. Alternatively, a gene sequence encoding the ligand can be inserted into the viral genome and represented on the surface of the virus particle. If desired, the site to which the virus particle binds can be transferred to different haptens, such as biotin and dinitrophenol (DNP), and to different virus particles cross-linked to anti-biotin and anti-DNP antibodies, allowing the virus particle to bind to the target itself. Alternatively, it is led to the binding site on the ligand. Therefore, the term "binding of a virus to a target substance" as used herein refers to a site where a virus directly or indirectly attaches to a target substance by any method and affects the target substance to maintain the activity of a related virus. To provide.

[0012] Proper pairing of the target substance, ie, virus and ligand, can be easily established using well-known methods and simple tests and error tests. The combination of potential viruses and ligands can form a variety of substances that can bind to a given target substance, and further different types of viruses can make various mutations. The present invention is not limited to bacteria as in the previously proposed virus amplification assay technology, and can form a target for virus binding with various target substances. Thus, the invention can be used extensively wherever it is desired to detect the presence of a substance in various samples.

[0013] The binding of the virus or mutant virus to the target substance is achieved using various methods and substances depending on the nature of the target substance. Generally, this conjugation occurs in one stage with a mixture of viruses in the presence of the appropriate virus, or at a site where virion binding is found only in one type of virus.
At one of the two stages, this is accomplished by culturing a sample containing the desired target material. The cultivation is generally carried out in a suitable liquid culture medium, especially a water-soluble culture medium, at a temperature of 0 to 60 ° C. The virus that is not bound to the target substance is in suspension in a solution or liquid phase, or in a solid foam, and is carried by a solid carrier such as nitrocellulose or a nylon membrane, a ceramic frit, a solid bead, or the like. The present invention will be described below for convenience in terms of the use of an aqueous carrier for the target material. The formation of such a target can be made by dissolving or suspending freeze-dried or other viral solids and target material by conventional techniques.

In order to form a third substance with both types of virus, two separate components interacting with each other, each having an independent virus type, are used in a particularly preferred embodiment. This is because rapid screening of drugs that affect the interaction is possible. Culture in stage a is performed until a sufficient proportion of the viral particles have bound to the target substance. The optimal conditions for the virus binding stage depend on the nature of the target material, the method of detecting the virus particles and the indicator attached to the virus in stage b. In general, virus binding takes 5 to 180 minutes at temperatures near 60 ° C., and the optimal duration and conditions are easily determined by simple tests and error tests on a case-by-case basis. The optimal range of viral binding to the target material depends on the nature of the properties detected in the output from stage b and the expected concentration of virus-bound target material in the sample, but in general, The binding amounts to at least 25% of the target material, preferably 30-60% or more.

If desired, the virus-bound target material formed in stage a
It can be isolated from the mixture by conventional isolation and washing techniques. However, this is often not necessary, and the output from stage a is used directly as the medium performed in stage b. However, the separation of half of the virus-bound target material from residual free virus particles can be achieved, for example, by filtration,
This is done by capturing with paramagnetic beads. Washing is performed with a substance that kills or disables residual free virus particles. Alternatively, where the binding of the virus particle to the target substance labels the target substance with a tag such as biotin, the virus-bound target substance may be labeled with a non-binding virus particle, such as a streptavidin paramagnetic bead. Isolated from. I have discovered that the separation or isolation of half of the virus-bound target material from such residual non-binding phage increases the sensitivity and specificity of the method of the present invention for detecting or determining the target material. did.

In stage B, the virus-bound target material is cultured in the presence of the indicator so that half of the virus is carried by the attachment of the target material. By these effects carried by one target molecule or particle,
Half of the virus attaches to the same indicator molecule or particle, yielding a molecule or particle with both of the two unique properties imparted to the target material. Thus, molecules or particles having both properties can be distinguished from molecules or particles having one property.

[0017] Half of the virus can attach to the indicator in a number of ways. For example, half of the virus will be infected with a bacterial indicator, or will genotype during culture in which the indicator is attached to impart specific properties thereto. However, the transfer of specific properties from the virus-bound target material to the indicator does not require entry of the virus particles into the cell of the indicator. In this way, the virus particles transfer their properties by attaching to the outside of the indicator.

For convenience, the present invention relates to a method wherein half of the virus carried by the target substance in stage b is infected to bacterial cells and protected in infected cells to confer specific properties on the infected cells. This is described below.

Thus, stage b requires the addition of an appropriate bacterial culture to the output of stage a and the conditions under which only those bacteria with specific properties capable of surviving or replicating are present. This is done by culturing and infecting bacteria. Thus, in a preferred embodiment, the cultivation of a bacterium, such as E. coli, is performed in the presence of two antibacterial agents, so that half of the virus carried by the virus-bound target confers resistance. Alternatively, the cultivation in stage b is performed in the absence of the antibiotic, and the antibiotic is added after the cultivation has been performed to provide a readily detectable bacterial population. For convenience, the present invention will now be described in terms of culturing bacteria in the presence of antibiotics so that the effects of bacterial cell growth can be detected in real time.

The E. coli is preferably present in quantities greater than those required to achieve a computational equivalence between the virus-bound molecule or particle and the bacterial cells, and is statistically required. An amount exceeding the amount is desirable. The use of an excess of E. coli reduces the chances of independent bacterial cells being infected by independent particles of both viruses that can remain in the output from stage a. However, stage b
Where medium or subsequent conditions strongly oppose the survival or growth of cells infected only with one type of virus, the use of excess amounts of E. coli may not be necessary. In general, the amount of E. coli that exceeds the statistical amount by 10 to 300% or more,
It is used to minimize the production of bacterial cells that are only infected with one type of virus, thus helping to detect cells that have been infected by the virus binding agent. Also, as noted above, it is desirable to remove unbound virus particles from the stage a product in order to further reduce the risk of producing bacterial cells that are only infected with one type of virus.

[0021] According to the effect of the antibiotic used in stage b, bacterial cells infected with only one virus may die or not grow or replicate. If a lytic gene virus is used, double infected cells can host virus replication. Thus, viral particles replicated from such cells can further infect and replicate cells so as to allow for a cascade of infection, replication and release of the viral particles. This has the effect of amplifying the growth effect of double infected cells.

[0022] The growth of double infected cells can be monitored using suitable techniques. For example, bacterial culture can be continued until an important population of double infected cells is visible to the naked eye. Alternatively, the growth of doubly infected cells is monitored by colorimetric, fluorescent, or chromogenic means, where one of the features provided with the target substance allows it, or where a third virus is bound to the target substance. can do. If desired, the culture means integrates enzymes or other means responsive to chemicals released from the growing bacteria to accentuate properties imparted by one of the viruses carried by the double infected cells. be able to.

The specific properties conferred on the double infected bacterial cells vary in intensity according to the number of such cells present in the stage b culture mixture. This intensity can be used to give an indication of the amount of target material in the original sample, as well as indicating the presence of target material in the initial sample tested. If desired, the effect of background ligation of the individual phage particles on the target substance and their detection in stage b can be achieved, in particular, using a detergent, for example a non-ionic detergent such as a solid under the trademark Tween, or Use of protein blocking agents such as casein in an amount of 0.1 to 0.5% v / v or albumin in an amount of more than 5% w / v in the culture medium or washing fluid in stages a and / or b Can be reduced by

As mentioned above, the method of the present invention can be applied to various target materials, depending on the number of combinations of materials that can be used. Thus, the present invention has a wide range of applications for various target materials in testing samples from various sources. The target substance need not be a living organism as required by phage amplification technology, but may be a chemical. For example, proteins derived from the lysis of viruses, RNs produced by living organisms rather than components of the organism itself.
A, heavy metals or other contaminants in the water supply system, or in solution or on a solid support such as a polyamide membrane.

The present invention aims to determine the biological activity of a substance by measuring the effect of the substance on inhibiting or promoting the growth of bacteria or other organisms. Thus, for example, certain organisms can be exposed to drugs for testing,
The production of hidden proteins or mRNA can be monitored. if,
If the drug is effective, it will stop producing protein or mRNA once the drug has taken effect. This is monitored by using appropriate techniques. Alternatively, the drug may inhibit the growth of a living organism, such as a fungus or yeast, and further growth of the organism after treatment of the drug evaluated in stage b.

The method of the present invention can also be used to study whether two or more substances affect each other in binding or reacting with each other. Each of the substances studied in the present invention can function as one target substance of the phage type, and both of the phage used to indicate that the two target substances interact or bind to each other occur. A product having a model can be produced. Thus, a molecule of one substance can be tagged with a phage having property A, and a molecule of the other substance can be tagged with a phage having property B. If the substance is exposed to the conditions in which interaction occurs and the resulting composition is cultured as in stage b, the presence of the molecule or product given property A and property B is detected as described above. can do. This technique can be applied, for example, in drug screening to detect the interaction of two single molecules and / or complex molecules, for example, the binding of enzymes on substrates, the interaction of two proteins or one protein with a nucleic acid. Can be. If desired, the third molecule can be conferred a third property using a different phage having a complex conferring all three properties of A, B and C. Alternatively, the present invention can demonstrate that a drug or other substance does not inhibit the interaction, for example, by preferentially binding with one of the interacting molecules, It can indicate whether the mixing of the conjugates of the substances is safe or whether the drug has the expected effect of the interaction.

In these cases, the presence or absence of the bound target substance formed by the interaction of the two separate components tagged by the virus, forming the target substance of the invention with two viral tags. The present invention is utilized for detecting Therefore, a target substance as used herein refers to one substance tagged with both types of viruses, and one substance tagged with one type and a substance that binds or influences each other to form a substance having both types of viruses. It is configured to contain substances that interact with each other.

[0028] In a preferred embodiment, detection of a target substance, since it includes the cultivation of co-infected cells, to the initial amount of cells is increased larger magnification, for example, 10 ⁸ to 10 times, the culture (Stage b) can be continued. In this case, very small levels of target material in the initial sample can be amplified to a very large range, which is the level of target material that would not have been detectable by conventional techniques.

The present invention also provides an analytical kit for use in the method, the kit comprising: a. Two phage substances that are compatible with a common target substance or individual components that form the target substance that bind to form the target substance and confer specific properties on the target substance; and b. An indicator suitable for culturing in the growth medium and adapted to adopt the specific properties conferred by the phage material. The indicator is preferably a bacterium that can be infected by the phage material.
More preferably, the kit also includes a growth medium for culturing the target substance and the indicator substance bound to the virus. It is further preferred that the kit comprises an additional component which allows a visual indication of the growth of the indicator substance infected by the phage substance.

[0030]

【Example】

Overview: The present invention is described below by way of example in terms of the preferred embodiment and in terms of detecting target molecules using two different viruses, virus A and virus B. The target molecule that will be bound in solution or on a support is likely a protein. Virus A and virus B are single-stranded bacteriophage M13 viruses or phagemids mutated by insertion of genes encoding ampicillin and kanamycin resistance, respectively. If desired, the virus can be mutated by insertion of a gene encoding an IgG linked to a region of the protein. This allows the virus to bind to any IgG antibody that can be identified against the target material. Where the target molecule is in solution, both or one of viruses A and B may be further mutated to include maltose bound to the peptide or autobiotinylated peptide, or covalently linked to a hapten such as biotin. Can be. This allows the virus-bound target material to be washed by capturing it with a streptavidin or merulose-inducible paramagnetic bead to remove unbound virus. This increases the sensitivity and specificity of the method of the invention.

The virus A and / or the virus B is a gene encoding a detectable marker such as an enzyme capable of detecting the colorimetric, fluorescent, and luminescent properties of the indicator attached to the virus, for example, β-galactosidase or luciferase. It can also be mutated to include If desired, virus A and virus B may each contain different components that are compiled for the detectable marker and function as dual infections of the indicator. A clear example of this is the display of β-galactosidase with LacZ complementation in TG1 cells. Expression of β-galactosidase only occurs on TG1 cells with virus-bound target material that has both complement components of the lac operon. β-
Galactosidase can be detected using the enzyme-derived source isopropyl-β-thiogalactopyranoside and the indicator bromo-4-chloro-3-indolyl-β-galactoside in an aged medium. The medium may include 4-methylumbelliferyl-β-D-galactose, which is fluorescent or chromogenic in the presence of β-galactosidase. Using a spectrometer, ELISA reader, luminometer or fluorimeter to monitor intermittent or continuously changing color or degree of color to obtain qualitative detection of the target substance present in the initial sample This is also included in the scope of the present invention.

A variation of this technique includes two interacting components for the formation of the third component, such that the third component has both phase types and can then be detected using the method of the present invention. The components can each be individually bound to different phases. This variation is a rapid detection of whether the activity of a drug that suppresses or enhances the production of a third component is likely to have a useful effect in the presence or occurrence of the first two components, eg, a protein. Provides a particular use for rapid screening of drugs where used to obtain

In the method of the invention, the virus particles are incubated with the sample in which the target molecule is detected at a temperature between 4 and 50 ° C. for 10 to 120 minutes. During this culture, the virus binds to the target protein molecules in the sample. After this culture, the sample is used or separated in culture stage b, for example for bead capture, and washed. The sample can be rinsed with a suitable wash buffer where it binds to the solid support.

In culture stage b, the amount of E. coli exceeding the statistical requirement is subsequently added to a suitable growth medium and incubated at a temperature between 4 and 50 ° C. for 30 to 720 minutes. Those infected with both viruses A and B are resistant to antibiotics and replicate, but the growth medium is ampicillin so that bacteria that have infected only one of the viruses are not killed or replicated. Contains both kanamycin. This allows the observer to monitor the growth of bacterial cells in the culture medium and to determine the presence of growing cells and their number in real time. However, it is also possible to add the antibiotic to the culture medium after an appropriate culture period and to determine the effect of the antibiotic on the cell population. If antibiotics were added after the initial culture stage, additional cultivation for between 2 and 60 minutes would be required to produce a detectable change in the culture material.

In another preferred embodiment for the detection of molecules, such as nucleic acids, in solution or bound to a solid support, virus A and virus B are single-stranded bacteriophage M13 virus or Phagemid mutated by insertion of genes encoding ampicillin and kanamycin resistance, respectively. The virus is susceptible to further mutation by binding to nucleic acids or by peptide nucleic acid probes as described in more detail below. The virus is present in the sample for 30-24 hours, along with the sample in which
Incubate for 0 minutes at a temperature between 4 and 60 ° C. During this culture, the virus binds to the target nucleic acid molecules in the sample. After this cultivation, the virus-bound nucleic acid is cultured in an appropriate growth medium at a temperature between 4 and 50 ° C. for 30 to 480 minutes in excess of E. coli and the effect of the antibiotic as described above. Is detected.

In the detection of nucleic acids, two or more nucleic acid probes capable of binding to a target nucleic acid are required. A single copy gene sequence can be detected by using two probes for the same gene region or gene, but the sensitivity of diagnostic applications depends on the number of targeted repeats in the genome of the organism being detected. Or by repeated sequences. Such repeated sequences are detected by the binding of two or more copies of a single type of probe to the repeated sequences. If two probes are used, they can be labeled with the same or different haptens. For example, both probes are labeled with biotin, or one probe is labeled with biotin and the other with digoxigenin (Boehringer Mannheim. Lewes, UK, 1 093 088). After binding (producing a hybrid) of the probe to the target nucleic acid, spatially bound probe molecules are detected by the hapten groups being carried by them. For example, phage A and phage K conjugated to a fragment of an anti-digoxigenin antibody (Boehringer Mannheim. Lewes, UK, 1214 667) or streptavidin can be used. If a single hapten, such as biotin, was used to label the probe, then 2
One phage can be labeled with the same hapten binding molecule, eg, streptavidin. This approach involves molecules that are repeated before and after repetition, such as the sequence Lmet2 of Leishmania donovani, or 21 base of Plasmodium falciparum.
Suitable for detecting pair repeats. Target DNA is prepared from the organism using appropriate conventional techniques or from natural extraction or by lysate of the sacrificial sample, and is applied to membranes such as Hybond N, Hybond N + (Amersham International plc, Amersham).
ham UK) and standardized for properties prior to detection (Amer
sham International plc publication P1 / 384/91/6 and P1 / 387/92/4 and Short Protocols in
Molecular Biology, 2nd edition Ausubel, FM et al eds. Green Publishing Associ
ates and John Wiley & Sons. 1992). Alternatively, a homogenous potency assay format can be used. Alternatively, purified DNA can be prepared, cut with restriction enzymes, separated in size by electrophoresis, and immobilized on a nylon membrane prior to detection.

Test Results: Preparation of Log Phase TG1 E. coli: Minimal media plates were prepared using FM Ausubel et al., Green Publishing Associates and Jo.
"Short Protocols in Molec" edited by hn Wiley & Sons in 1992
TG1 E. coli (Pharmaci).
a Biotechnology, UK-Expression Module 27-9401-01), streaked on minimal media plate, cultured overnight at 37 ° C to generate single colonies, then left at 4 ° C. A single colony is 17 g / l sterilized for 15 minutes
Bactotryptone (Difco 0123-17-3), 10g Bacto yeast extract (Difco
0123-17-19) and 10 g of 2 × YT medium containing 5 g of NaCl. The inoculation medium was shaken at 37 ° C. until a log growth of OD 660 nm of about 0.5 was achieved.

Preparation of phage K: 10 μl of Logphase TG1 E. coli prepared as described above was added to 1 ml of 2 × YT in a 20 ml plastic universal container. 10 ⁸ phages
M13K02 (Pharmacia, Expression module 27-1524-01) pfus was added to the vessel and the mixture was incubated at 37 ° C. for 60 minutes. 2 x YT 10 ml, kanamycin 50 μg /
Then, the cells were mixed and cultured at 37 ° C. for about 5 hours until steady phase growth was achieved. Cells and cell debris were removed by centrifugation and the supernatant filtered through a 0.22 micron filter. 2 mls of a 20% w / v solution of PEG 8000 (Sigma P5413) and 2
. 5M NaCl was added to the phage suspension and the phage was precipitated overnight. The phage was then re-precipitated by repeated centrifugation and then PBS (S
igma 1000-3) 1 ml, MgCl ₂ (Sigma M2670) 1 mM and trypsin 25 μM
(2.5% wv Trypsin stock-Sigma T2670, as 6 μl) for 60 minutes, and the phage was precipitated in 200 μl of 20% PEG8000 and 2.5 M NaCl for 10 minutes at room temperature. The phage is centrifuged, and PBS, MgCl ₂ , PEG8000 and N
Washed with aCl. The liquid was removed and the phage was reprecipitated to obtain active phage with the kanamycin genome.

Preparation of phage A: This phage was prepared using Pharmacia Biotechnology, Expression module 27-9401-01.
Using phage A pCANTAB 5E from E. coli and ampicillin (Sigma A2804).
Phage except that it was cultured with the helper phage M13K07 from Pharmacia Biotechnology, Expression module 27-1524-01 and then with both ampicillin and kanamycin (Sigma K0879) to obtain a phagemid encoding ampicillin resistance. K was prepared using the technique described above. Alternatively, phage A and phage K can be prepared and purified by ultracentrifugation on a cesium chloride gradient using the techniques described in Short Protocols of Molecular Biology.

Example 1 Detection of Immobilized Ligand Binding Molecules Using Ligand Labeled Phage The ligand used in this example is biotin which is bound to the protein streptavidin. One molecule of streptavidin can bind to four biotin molecules. Biotinylation of phage was performed using biotinamide caproate-N-hydroxysulfosuccinimide ester (Sigma B2643) as described in the manufacturer's handbook.
). The optimal ratio of phage to biotin can be determined experimentally for each phage. Biotinylated phage was purified by standard polyethylene glycol precipitation.

[0041] Biotinylated phage K and phage A were used to detect streptavidin paramagnetic beads. Use a hemocytometer for this bead.
2 × 10 ⁵ particles per weight were previously weighed by light microscopy. Streptavidin paramagnetic beads (Promega Z5481) were washed and prepared as recommended by the supplier. Control beads were 0.1 mM biotin (Sigma B4501
) Was blocked by washing in the same solution containing Streptavidin and blocked control beads were serially diluted 10-fold and cultured with each mixture of biotinylated phage. After binding the phage to the beads, the beads are washed and the log phase T
Cultured with G1 cells.

After phage infection, TG1 cells were stuck to a solid medium containing ampicillin and kanamycin to selectively culture E. coli cells that had become cells double infected by both phage A and phage K. And the number of colonies was counted (see Table 1). Tests and blocked controls are duplicated (A and B),
The results are shown in Table 1.

[Table 1]

Conclusion: Two phages encoding different selectable properties within their E. coli host can be used in assays to detect molecules bound by the potency of ligands on the phage surface. it can. The results demonstrate that this test method has a sensitivity as low as 20 streptavidin beads.

Example 2 Detection of Immobilized Ligand Binding Molecule (Streptavidin) in Homogeneous Assay (No Wash) Serial dilutions of streptavidin (Sigma S4762) were prepared using biotinylated phage A prepared as in Example 1. Added to the mixture of phage K. After binding the phage to streptavidin, log phase TG1 cells were added along with the phage to infect E. coli cells.

After infection, the cells were plated on a solid medium containing ampicillin and kanamycin to select for double infected cells, the number of colonies on each plate was counted, and the results are shown in Table 2.

[Table 2]

[0046] Conclusion: These results, the method of the present invention, without complex capture and washing, 10 ⁵ molecules or
It has been demonstrated that detection is as small as 0.1 attomoles streptavidin. Inhibition of detection is evident above streptavidin concentrations (1 femtomole), a common observation with other immunoassay techniques.

Example 3 Detection of Antigen by Antibody-Labeled Phage Phage A and phage K were subjected to Bioconjugation, Mohammed Aslam and Alastair.
Dent, Eds. 1998. Macmillan Reference Ltd. London, UK ISBN 0-333-583752, by using glutaraldehyde as an anti-potato virus X antibody (Agdia Incorporated, Elkhart, Indiana 46514, US
A). Dilute the conjugate and place in 1 mM MgCl ₂ TBS at 4 ° C.
Stored at ELISA kit for detection of potato virus X antibody (Agdia PathoScreen Kit PSP 10000/0288), positive control vial of freeze-dried infected leaf material (Agdia, LPC 10000) and negative of freeze-dried leaf material Control vials (Agdia, LNC 10000) were obtained from Agdia.

Dilutions of infected leaf material were added to anti-potato X virus antibodies coated on microplate wells (Agdia, PathoScreen Kit PSP 10000/0288) to capture viral material. After capture and washing, conjugated phage A and phage K
Was added to each well and cultured to capture phage particles. Wells were then washed and log phase TG1 cells were added along with phage conjugated to allow infection of the cells. After infection, the contents of each well were applied to a solid medium containing ampicillin and kanamycin to select for double infected cells, and the number of colonies on each plate was counted. Table 3 shows the results.

[Table 3]

Conclusion: The method of the present invention detected the presence of leaf virus in a 10 ^-3 dilution of the positive control leaf material.

COMPARATIVE EXAMPLE ELISA approach for comparison of one phage, two phage, and detection of virus A single conjugated phage (phage K) for detection of diluted plant extracts
Example 3 except that the use of was compared to a mixture of conjugated phage A and phage K.
The same protocol was followed as described in The single phage K was finally plated on agar plates containing kanamycin, whereas the phage mixture was finally plated on agar plates containing kanamycin and ampicillin in the usual way. After culturing to select for infected cells, the number of colonies was counted and the results are shown in Table 4.

The Pathoscreen potato virus X ELISA assay was performed in parallel with the same dilution of the positive control leaf extract according to the manufacturer's instructions, and the results of this test are also shown in Table 4.

[Table 4]

Conclusion: This experiment demonstrates that it is advantageous to use a two phage approach as compared to a single phage approach. When used with phage K alone, a large number of colonies were present in the negative control. The two phage approach reduces the background signal so that positive samples are easily distinguished even at a dilution of 10 ^-3 . The background signal is generated by the use of detergents, such as solids of the trademark Tween 20 at a concentration of 0.1-0.5% v / v, and / or bovine serum albumin or a washing medium at a concentration of up to 5% w / v in the culture. It can be further reduced. The PathoScreen ELISA supplied by the manufacturer was not as sensitive as the method of the present invention.

Example 4 Alternative Two-Phage Strategy for Virus Detection Anti-Potato X virus conjugated with phage A or phage K (prepared as in Example 3) was added to microplate wells (Greiner Labortechnik Ltd., Cambridg
e, UK, 756061). Dilutions of the plant extract were captured in phage-coated wells. After washing, the phage / antibody conjugate (phage A or phage K) is added back to the wells where phage K was coated with phage A, such that phage A was added to the wells coated with phage K. Was added to each well as described. After culturing to capture phage, wells were washed and added to infect log phase TG1. After infection, the contents of each well were applied to a solid medium containing ampicillin and kanamycin to select for double infected cells. After selection, the number of colonies on each plate was counted, and the results are shown in Table 5.

[Table 5]

Conclusion: This test shows that virus binding to the target material in two stages can effectively replace the binding of phage particles in one stage, and 10 ⁻⁴ of the positive control leaf extract It has been demonstrated that detection with dilutions is possible.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR , BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS , JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZWF terms (reference) 2G045 BB14 BB20 BB29 CB21 FA16 FA18 FB01 FB03 GC22 4B063 QA01 QA20 QQ05 QQ06 QQ07 QQ08 QQ10 QQ21 QQ41 QQ61 QQ70 QQ79 QQ89 QQ91 QQ94 QQ96 QQ98 QR15 QR54 QR75 QR79 QR83 QS24 QX01

Claims (20)

[Claims] The present invention relates to a method for identifying a target substance in a sample, the method comprising the following steps: a) a method in which a sample expected to contain the target substance or concentrated in the target substance is subjected to virus binding; Exposing to two or more viruses that can bind directly or indirectly to the target material to obtain a virus-bound target material having specific properties and forming the target material; b) from stage a. Culturing the product in the presence of the indicator to a virus carried by the attachment of the virus-bound target material such that the indicator can accept the specific properties of the virus-bound target material; andc. ) Monitor the presence or absence of the virus attached indicator. 2. The method according to claim 2, wherein the culture stage b is carried out in a state where the indicator to which the virus has adhered survives, but the indicator to which only the virus has adhered or the virus has not adhered at all does not survive. The method according to claim 1, wherein 3. The method according to claim 1, wherein half of the virus is bound to the target substance via an intermediate ligand. 4. The method according to claim 1, wherein half of the virus imparts two different forms of the antibiotic to the virus-bound target substance. 5. The method according to claim 1, wherein the stage b is cultured in the presence of the indicator bacteria. 6. The method according to claim 5, wherein the culture of stage b is performed in the presence of at least a statistically required amount of bacterial cells. 7. The method according to claim 1, wherein a virus having a visually detectable characteristic is used as the virus infection indicator. 8. The method according to claim 1, wherein the target substance is an inorganic or organic substance, a living body, a part or a symbol thereof, or a product of a living body. 9. The target substance is a protein, an enzyme, a prion, a lipid, a nucleic acid,
9. The method according to claim 8, wherein the method is a hormone, fungus, virus, bacterium, or protozoa. 10. The method of claim 1, wherein the target material is the result of an interaction of two or more materials, and the method is used to determine the suppression or limit of such interaction. 10. The method according to any one of claims 1 to 9. 11. The method according to claim 10, wherein the interaction is suppressed by the action of the third component, and the effect of the third component is assayed depending on the degree of suppression of the product of the interaction. The described method. 12. The method of claim 1, wherein the target material is a contaminant or impurity present in another material, and the method is used to detect the presence of such a contaminant or impurity. The described method. 13. The method according to claim 13, wherein the target substance is a drug or a biocide, or a biological biocide or an interaction product of the drug, wherein the method is used to detect the effect of the drug or the biocide. The method according to claim 1, wherein the method comprises: 14. The method of claim 1, wherein the detection of a specific property in the indicator is monitored over a period of time to provide a quantitative indication of a target present in the sample. The described method. 15. A method substantially as claimed in claim 1 as described in any aspect of the preferred embodiment prior to this claim. 16. At least two phage substances suitable for binding to a common target substance or a component that interacts with the target substance and imparts specific properties to the target substance; And b) an indicator suitable for accepting the specific properties conferred by the phage material and suitable as a medium for the growth medium, a kit of components for use in the method of claim 1. 17. The kit according to claim 16, wherein the indicator substance is a bacterium that can be infected by a phage substance. 18. The kit according to claim 15, wherein the phage substance and the bacterium are in a specific form of a freeze-dried solid. 19. The kit according to claim 16, further comprising a growth medium culture for culturing a virus-bound target substance and an indicator substance. 20. The kit according to claim 16, further comprising an additional component for providing an indicator capable of visually detecting the growth of the indicator infected by the phage substance.